Project Summary Retinal ganglion cells (RGCs) from each eye extend ipsi- and contralaterally to the brain to establish the circuit for binocular vision. Progress since the beginning of this grant has led to the identification of transcriptional regulators that contribute to the specification and differentiation of these two RGC subtypes, ultimately, to differential axon crossing at the midline. Three new paths have arisen from this work: First, a switch from emphasis on neurogenesis that gives rise to the central part of the retina to the analysis of neurogenesis in the ciliary marginal zone (CMZ), a peripheral progenitor cell population that until recently was thought not to be a major source of retinal neurons in mammals, as is the case in lower vertebrates. Our recent study demonstrated that cells translocate tangentially from the CMZ to the neural retina and thus the CMZ is posited as a neurogenic source of RGCs during embryogenesis. We will use genetic fate mapping and retinal cell and RGC subtype marker expression to determine the identity of cells emanating from the CMZ and their projections. Because the CMZ is adjacent to the ventrotemporal retina (VT) from which the ipsilateral RGCs arise, we will pay special attention to the production of ipsilateral RGCs, but also chart contralateral RGCs that are arise from VT retina later in development (Aim 1). Second, Cyclin D2 as a regulator of RGC genesis in the CMZ: In other brain areas, Cyclin D2 is expressed in neuronal subpopulations and through regulating cell cycle, is thought to regulate cell fate, but its mechanism of action is poorly understood. Our work reveals that Cyclin D2 is enriched in the ventral CMZ. Moreover, in the absence of Cyclin D2, neurogenesis of ipsi and contra RGC subtypes is reduced and the ipsilateral RGC projection decreased. The mechanism of Cyclin D2 in these processes. particularly cell fate, cell translocation, and cell cycle, will be analyzed in mice lacking Cyclin D2 (Aim 2). Third, the genes that pattern the CMZ such as the Wnts and their relevance to proper production of ipsi- and contralateral RGCs: Through loss-of-function models of this pathway, starting with the beta-catenin conditional knockout, the CMZ and the organization of the Cyclin D2?expressing zone, and RGC subtypes produced in the neural retina will be charted (Aim 3). Thus, the goal of this renewal application is to further characterize the RGC neurons generated in the CMZ, with specific attention to their ipsi- or contralateral projection (Aim 1), to understand how CyclinD2 regulates the generation of RGCs in the CMZ (Aim 2), and to dissect how patterning of the CMZ by known morphogens impacts the generation of RGCs and their projections (Aim 3). Significance: Identifying gene programs on how ipsi-contra diversity arises will elucidate how decussating systems such as the binocular circuit are established. Such information is critical for driving stem cells into RGCs for replacement therapy and directing axon regeneration in injured and degenerating visual pathways.